June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Cathepsin D deficiency in RPE cells mediates Stargardt Disease pathogenesis
Author Affiliations & Notes
  • Eunice Sze Yin Ng
    UCLA Stein Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Jane G Hu
    UCLA Stein Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Zhichun Jiang
    UCLA Stein Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Michael B Gorin
    UCLA Stein Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Anna Matynia
    UCLA Stein Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Roxana A Radu
    UCLA Stein Eye Institute, Los Angeles, California, United States
    Ophthalmology, University of California Los Angeles David Geffen School of Medicine, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Eunice Sze Yin Ng, None; Jane Hu, None; Zhichun Jiang, None; Michael Gorin, None; Anna Matynia, None; Roxana Radu, None
  • Footnotes
    Support  Stein Eye Institute Core Grant for Vision Research NIH Grant EY000331, Vision Research Training Grant NIH Grant 5T32EY7026-43, Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology at UCLA Stein Eye Institute
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 3113. doi:
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      Eunice Sze Yin Ng, Jane G Hu, Zhichun Jiang, Michael B Gorin, Anna Matynia, Roxana A Radu; Cathepsin D deficiency in RPE cells mediates Stargardt Disease pathogenesis. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3113.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : In the retinal pigment epithelium (RPE), Cathepsin D (CatD) is the principal lysosomal protease responsible for breaking down internalized proteins. CatD activity and its maturation through the endolysosomes requires a low pH. Key pathological hallmark of Stargardt disease (STGD1) and its mouse model (Abca4-/-) is the accumulation of autofluorescent lipofuscin in the RPE. Both STGD1 donor eye and Abca4-/- RPE showed significant deposition of complement proteins on the RPE cells. These RPE lipofuscin granules contain A2E-bisretinoids, lipid and protein aggregates, including complement fragments. Notably, increased lysosomal pH in the RPE was previously evidenced in the Abca4-/- mice. In this study, we investigated the specific role of CatD in the endolysosomes of STGD1 experimental models.

Methods : Age-matched wild-type (WT) and Abca4-/- mice (n=3) were used to prepare RPE homogenates for immunoblotting analysis of mature CatD level. Pooled RPE sheets of 6-mo-old WT and Abca4-/- mice (n=4) were used for proteomics analysis by liquid chromatography-mass spectrometry (LC-MS). Functional activity of CatD in RPE cells of WT and Abca4-/- mice (n=3) were measured by fluorometric assay. Statistical significance was determined by paired t-test. CatD and α-synuclein, its physiological substrate, were evaluated by immunohistochemistry (IHC) in fixed mouse eyes (WT and Abca4-/-) and induced pluripotent stem cell (iPSC) derived RPE cells from STGD1 patient vs control unaffected individuals.

Results : By LC-MS proteomics analysis, we found a ~1.6-fold (p<0.001) increase in level of total CatD (immature, intermediate, mature) forms in RPE cells of Abca4-/- mice vs WT. Surprisingly, quantitative immunoblotting analysis showed a ~1.7-fold (p=0.042) decrease of mature CatD in the RPE of Abca4-/- mice vs WT. Importantly, CatD functional activity was reduced in the RPE of Abca4-/- mice by ~42% (p=0.024) compared to WT. By IHC, STGD1 iPSC-RPE cells but not normal controls showed significant accumulation of α-synuclein consistent with impaired CatD activity.

Conclusions : Our studies suggest that CatD is an integral component of maintaining the homeostasis of the RPE cells. In STGD1 RPE cells, decreased CatD functional activity and reduced protein maturation further exacerbate the endolysosomal dysfunction. Our findings introduce a molecular mechanism involving CatD in the pathophysiology of STGD1 with important therapeutic implications.

This is a 2021 ARVO Annual Meeting abstract.

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